PROJECT SUMMARY IL-10 producing type 1 regulatory T cells (Tr1 cells) play an important role in controlling the immune response. Thus, Tr1 cells are potential targets for treating inflammatory disorders, but little is known about the control of their differentiation, stability and function. We have found that the transcription factor aryl hydrocarbon receptor (AHR) promotes Tr1 cell differentiation. AHR drives the expression of target genes in Tr1 cells, for example Entpd1 which codes for CD39. CD39 degrades extracellular adenosine triphosphate (eATP) and synthesizes immunosuppressive adenosine. We now found that: 1) eATP and hypoxia, which characterize inflamed tissues, inhibit Tr1 cell differentiation. 2) eATP and hypoxia activate the hypoxia- inducible factor-1? (HIF1?), which inhibits AHR-driven gene expression. 3) CD39 expressed in Tr1 cells (CD39Tr1) promotes Tr1 cell differentiation by depleting eATP. 4) CD39Tr1 deficiency impairs Tr1 cell differentiation and suppressive function. 5) CD39 expressed in DCs (CD39DC) promotes Tr1 cell differentiation. These findings suggest that CD39 controls Tr1 cell dependent immune regulation by stabilizing AHR-driven gene expression. We hypothesize that the balance between AHR/CD39 and eATP/HIF1? controls Tr1 cells. The objective of this project is to elucidate the role of these novel pathways on Tr1 cells, and their relevance for multiple sclerosis (MS) and other autoimmune diseases. Our specific aims are: Specific Aim 1: What is the role of CD39Tr1 in Tr1 cell stability and function? CD39Tr1 deficiency impairs AHR-driven gene expression and suppressive function in Tr1 cells. These data suggest that by depleting eATP, CD39Tr1 stabilizes the AHR-driven transcriptional program of Tr1 cells. Thus, we propose to: 1) Define the role of CD39Tr1 on Tr1 cell stability during experimental autoimmune encephalomyelitis (EAE). 2) Determine the effects of eATP and hypoxia on Tr1 cell stability and their control by CD39Tr1. 3) Establish the effects of eATP and hypoxia on the transcriptional program of Tr1 cells. Specific Aim 2: Does CD39Tr1 control human Tr1 cells? The roles of AHR/CD39 and eATP/HIF1? on human Tr1 cells and their therapeutic potential are unknown. Thus, we propose to: 1) Establish the roles of AHR/CD39 and eATP-Hypoxia/HIF1? on the differentiation of human Tr1 cells. 2) Determine whether deficits in CD39+ Tr1 cells are associated to MS. Specific Aim 3: How does CD39DC control Tr1 cells? CD39DC promotes IL-10 expression by T cells in vitro and limits autoimmune inflammation. Thus, we propose to: 1) Determine the role of CD39DC in controlling Tr1 cells in vivo. 2) Establish the role of eATP signaling in T cells on the control of Tr1 cells by CD39DC. 3) Define the effects of CD39DC on DC phenotype and function. IN SUMMARY, we use unique tools in mouse and human systems to study novel pathways that control Tr1 cells, as well as potential therapeutic targets for MS and other autoimmune diseases. 2